Can I perform in-vitro transcription with the pDrive Cloning Vector from the QIAGEN PCR Cloning Kit?
Yes, it is possible to use the pDrive Cloning Vector from the QIAGEN PCR Cloning Kit for in-vitro transcription. It contains SP6 and T7 polymerase promoters. However, we do not have a special protocol for this purpose. We recommend to varify insert orientation via sequencing. Note that the pDrive Cloning Vector does not contain transcription terminators. Therefore, we recommend to linearize the vector at the end of the insert to create a stop point for RNA Polymerase. To overcome the very high template affinity of this enzyme, use a cutter that provides a 5' overhang, thereby promoting that it falls off the template. The T7 transcription start site is located within the promoter region, leading to transcription of the last three guanidine bases of the promoter. When planning to use the in-vitro transcript as a standard for RT-PCR, note that it will also contain truncated products.